In FY 91, the Genetic Pharmacology Unit focused on the molecular regulation of striatal dopamine receptor genes and of a model system for peptide transmitters: 1. Transcriptional regulation of the Dl dopamine receptor gene. This project which was initiated at the end of FY 90 involves three main series of experiments. First, the human adenylate cyclase-linked Dl gene has been cloned including its 5' regulatory elements, the nucleotide sequence of a 2.4 kb stretch upstream from the known sequence has been determined and found to lack TATA and CAAT boxes and to be rich in GC content. In addition, a transcription initiation site has been identified and cell type-specific enhancer/promoter activity of several deletion mutants of the 5' flanking region delineated. Second, the transcriptional regulation of the mouse Dl gene endogenously expressed in a neural cell line is being studied in response to various pharmacologic stimuli. Third, mRNA coding for adenylate cyclase-linked Dl receptor was identified, for the first time, in renal tissue. 2. Promotor analysis of the rat D2 dopamine receptor gene. Also initiated during the last quarter of FY 90, the gene coding for the rat D2 receptor is now successfully cloned which encompasses the 5' upstream elements based on sequence information and determination of transcription start site. Like the human Dl gene, the rat D2 gene lacks a TATA box and a CAAT box. Thus, both the Dl and D2 receptor genes belong to the category of tissue-specific TATA-lacking genes. The promoter/enhancer activity of various fragments of the D2 5' untranscribed region is currently being characterized. 3. Regulation of POMC gene transcription. Studies done in FY 91 were based on earlier findings in this laboratory about two protein-binding DNA elements in the 5' upstream region of the mouse POMC gene: one located between -119 and -106 bp relative to transcription start site and is highly homologous to AP-2 consensus sequence, and the other located between bases -137 and -131 and has a 70% homology to, but is distinct from, AP-1 consensus sequence. Because of the physical proximity of these two protein-binding elements, their potential interaction in modulating gene transcription is currently being investigated using the reporter gene CAT.